WO2020238179A1 - Pyrrolopyrimidine compound as selective jak2 inhibitor, synthesis method therefor and use thereof - Google Patents

Abstract

The present invention belongs to the technical field of biological medicine, and specifically relates to a pyrrolopyrimidine compound as a selective JAK2 inhibitor, a synthesis method therefor and the use thereof. Compared with the prior art, the pyrrolopyrimidine compound provided by the present invention, a stereoisomer thereof and a pharmaceutically acceptable salt thereof have a better Janus Kinase inhibitory activity, and the selectivity thereof for JAK2 inhibitory targets is obviously better than that of an existing compound, and the preferred compound of the present invention exhibits a good pharmacokinetic property and has the potential to be developed as a selective JAK2 inhibitor.

Description

Pyrrolopyrimidine compounds as selective JAK2 inhibitors, synthesis methods and uses thereof Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to a pyrrolopyrimidine compound as a selective JAK2 inhibitor, its synthesis method and application.

Background technique

JAK stands for Janus Kinase, and it is a non-receptor tyrosine protein kinase (PTK). JAK-STAT pathway is mainly composed of four parts: (1) extracellular signal factor; (2) receptor; (3) JAK kinase; (4) signal transduction and activation protein of transcription (STAT). JAK-STAT is the most important signal pathway besides the second messenger system. JAK kinase senses extracellular signals, such as interferons, interleukins, growth factors, etc., by binding to receptors, and transmits information to STATs. Phosphorylated STATs can be transferred from the cell to the nucleus. Each different STAT binds to a different promoter DNA sequence. Promoters control the expression of their DNA sequences, causing changes in DNA transcription and activity levels, which in turn affect basic cell functions such as cell growth, differentiation and death.

There are 4 proteins in the JAK kinase family, including JAK1, JAK2, JAK3, and TYK2. From the perspective of gain-of-function expression or mutation analysis, JAK1 and JAK3 are more related to immune regulation, while JAK2 is directly related to the production of red blood cells and platelets. From the perspective of functional loss analysis, the loss of JAK1 and JAK2 functions can cause the death of mouse embryos. No diseases related to the loss of JAK1 and JAK2 functions have been found in humans, which may indirectly indicate the importance of the physiological functions of JAK1/2. The lack of JAK3 function can cause serious comprehensive immune deficiency, which is also the basis for targeting JAK3 mentioned later to regulate autoimmune-related diseases. There are few studies on the function of TYK2, and it has been reported that it can cause defects related to intrinsic immunity.

The discovery of JAK2V617F mutation in myeloproliferative tumors (MPN) has greatly promoted the development of JAK2 inhibitors. MPN is a group of chronic diseases characterized by the proliferation of abnormal hematopoietic progenitor cells in the bone marrow. MPN includes myelofibrosis (MF), polycythemia vera (PV), essential thrombocythemia (ET), and chronic myelogenous leukemia (CML). Approximately 95% of PV patients and 50-60% of MF and ET patients have been found to have JAK2V617F single amino acid mutations, which cause a conformational change of JAK2, resulting in the continuous activation of the kinase region independent of extracellular cytokine signals, which in turn causes cell proliferation And blood cancer.

Ruxolitinib reported in WO2007070514A was originally developed by Incyte and is a JAK1/JAK2 small molecule kinase inhibitor. It was approved by the FDA in November 2011 for the treatment of medium and high-risk myelofibrosis MF. It was further approved for polycythemia vera in 2014. Ruxolitinib can relieve the enlargement of the spleen caused by the JAK2V617F mutation and reduce the symptoms of weakness in patients.

Ruxolitinib cannot reduce the JAK2V617F mutation load of mutant blood cancer cells, so Ruxolitinib can hardly bring about a cure. In addition, due to the low selectivity of the JAK2 target of Ruxolitinib, the side effects are obvious. The side effects of Ruxolitinib mainly include anemia, thrombocytopenia, neutropenia and diarrhea.

Early reports showed that obvious and poor prognostic inflammatory syndrome occurred after Ruxolitinib was discontinued. In the subsequent 3 years of follow-up, no continuous similar adverse reactions were observed, suggesting that such reactions may be caused by discontinuation of Ruxolitinib For severe withdrawal inflammatory syndrome, the size of the spleen should be closely monitored. If the spleen still grows during Ruxolitinib treatment, the MF-related symptoms may return to the baseline level or even continue to progress after stopping the drug. Therefore, when considering interrupting Ruxolitinib treatment, the dose should be gradually reduced or corticosteroids should be used in combination.

The development of a new generation of MPN drugs focuses on selective inhibitors of JAK2, which is expected to reduce the excessive side effects caused by targeting JAK1 while increasing the efficacy.

At present, a series of patent applications for JAK inhibitors have been published, such as WO9965909A, WO2013173720A, WO2010039939A, WO2018019222A, WO2018087202A, etc. Although a series of JAK inhibitors have been published, there is still a need to develop new JAK inhibitor compounds with better efficacy and lower side effects, especially JAK2 selective inhibitors.

Summary of the invention

In order to overcome the problems in the prior art, the purpose of the present invention is to provide a selective inhibitor of JAK2.

In order to achieve the above objectives and other related objectives, the present invention adopts the following technical solutions:

A pyrrolopyrimidine compound as shown in formula I, its stereoisomers, and its pharmaceutically acceptable salt:

among them:

R ₁ is a C _1-6 alkyl group or a C _1-6 alkyl cyano group; the C _1-6 alkyl cyano group means that any hydrogen on the C _1-6 alkyl group is replaced by a cyano group;

R ₂ and R ₃ are each independently hydrogen or C _1-6 alkyl;

R ₄ is C _1-6 alkyl, -C(O)R ₅ , -C(O)NR ₆ R ₇ , -S(O) ₂ R ₈ or -S(O) ₂ NR ₉ R ₁₀ ;

a is 0, 1 or 2;

b is 0, 1 or 2;

c is 0, 1 or 2;

d is 0, 1 or 2;

R ₅ is a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group or a C _1-6 alkyl cyano group. The C _1-6 alkyl cyano group refers to a C _1-6 alkane Any hydrogen on the group is replaced by a cyano group;

R ₆ and R ₇ are each independently hydrogen, C _{1 ~ 6} alkyl group, C _{3 ~ 6} cycloalkyl, or R _6, R ₇ together with the N atom form a 3-6 membered ring;

R ₈ is C _1-6 alkyl or C _3-6 cycloalkyl;

R ₉ and R ₁₀ are each independently hydrogen, C _{1 ~ 6} alkyl group, C _{3 ~ 6} cycloalkyl, or R _9, R ₁₀ together with the N atom form a 3-6 membered ring.

Preferably, the R ₁ is a C _1-4 alkyl group or a C ₁ -C ₄ alkyl cyano group; the C ₁ -C ₄ alkyl cyano group means that any hydrogen on the C ₁ -C ₄ alkyl group is replaced by one Cyano substitution;

R ₂ and R ₃ are each independently hydrogen, methyl or ethyl;

R ₄ is C _1-6 alkyl, -C(O)R ₅ , -C(O)NR ₆ R ₇ , -S(O) ₂ R ₈ or -S(O) ₂ NR ₉ R ₁₀ ;

a is 0, 1 or 2;

b is 0 or 1;

c is 0, 1 or 2;

d is 1 or 2;

R ₅ is a C _1-4 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group or a C _1-4 alkyl cyano group, and the C _1-4 alkyl cyano group refers to a C _1-4 alkane Any hydrogen on the group is replaced by a cyano group;

R ₆ and R ₇ are each independently hydrogen, C _1-4 alkyl, C _3-6 cycloalkyl, or R ₆ , R ₇ and the connected N atom together form a 3-6 membered alicyclic ring;

R ₈ is C _1-5 alkyl or C _3-6 cycloalkyl;

R ₉ and R ₁₀ are each independently hydrogen, C _{1 ~ 6} alkyl group, C _{3 ~ 6} cycloalkyl, or R _9, R ₁₀ together with the N atom form a 3-6 membered aliphatic ring.

More preferably, said:

R ₁ is methyl, ethyl or

R ₂ and R ₃ are hydrogen;

R ₄ is C _1-3 alkyl, -C(O)R ₅ , -C(O)NR ₆ R ₇ , -S(O) ₂ R ₈ or -S(O) ₂ NR ₉ R ₁₀ ;

a is 0, 1 or 2;

b is 0 or 1;

c is 1 or 2;

d is 1;

R ₅ is C _1-3 alkyl, C _3-6 cycloalkyl,

R ₆ and R ₇ are each independently hydrogen, C _1-3 alkyl, C _3-6 cycloalkyl, or R ₆ , R ₇ and the connected N atom together form a 5-6 membered aliphatic ring; specifically, 5 The -6 membered alicyclic ring is tetrahydropyrrole or hexahydropyridine;

R ₈ is a C _1-5 alkyl group or a C _4-5 cycloalkyl group;

R ₉ and R ₁₀ are each independently hydrogen, C _1-3 alkyl, C _3-5 cycloalkyl or R ₉ , R ₁₀ and the connected N atom together form a 5-6 membered aliphatic ring; specifically, 5 The -6 membered aliphatic ring is tetrahydropyrrole or hexahydropyridine.

Furthermore, the stereoisomer is a stereoisomer formed by the C atom directly connected to R ₁ .

Furthermore, the pyrrolopyrimidine compound, its stereoisomers, and pharmaceutically acceptable salts thereof as shown in formula I:

Specifically, the pyrrolopyrimidine compounds provided by the present invention are shown in the following table:

The second objective of the present invention is to provide a synthetic method of the above compound:

(1) Compounds of general formula IA are prepared by Wittig reaction to compounds of general formula IB;

(2) Compound IC of general formula is prepared by catalyzed coupling reaction between IB and commercial compound 2;

(3) IC is deprotected and condensation reaction is used to prepare IE;

(4) Compound IE is deprotected to obtain the final product I; in the above reaction formula, the P1 group is the N protecting group; the X group is the activating group;

The P1 group is a C _1-6 alkoxycarbonyl group, which is tert-butoxycarbonyl in some embodiments of the application; specifically, the X group is selected from halogen, C _1-3 alkoxy or C _1-3 alkanesulfonic acid group, in some embodiments of this application, the X group is halogen.

The third object of the present invention is to provide the use of the above compound as a novel JAK inhibitor in the preparation of drugs for the prevention or treatment of JAK-related diseases. Specifically, it mainly refers to the prevention or treatment of the following diseases: diseases of the immune system, including organ transplant rejection (Such as allogeneic rejection and graft-versus-host disease); autoimmune diseases, including, for example, lupus, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, psoriasis, ulcerative colitis, Crohn’s Diseases, autoimmune thyroid diseases, etc.; skin diseases, including, for example, psoriasis, itching, atopic dermatitis, etc.: allergic diseases, including, for example, asthma, rhinitis, etc.; viral diseases, including, for example, hepatitis B and C Hepatitis, varicella-zoster virus, etc.; Type I diabetes and diabetic complications; Alzheimer's disease, dry eye, myelofibrosis, thrombocytosis, polycythemia or leukemia, multiple myeloma; cancer, including, for example Solid tumors (such as prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, melanoma, etc.), skin cancer (such as skin T cells) Lymphoma, skin fetal cell lymphoma), etc.

In the course of disease treatment, the derivatives of the present invention can be formed into a composition by oral, injection, etc., to treat related cancers and other diseases. When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders or capsules; when used for injection, it can be prepared into injections.

The fourth object of the present invention is to provide a composition comprising a therapeutically effective amount of the above-mentioned pyrrolopyrimidine compound, its stereoisomer, its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable salts, for example, metal salts, salt salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. can be mentioned. Non-limiting examples of metal salts include, but are not limited to, alkali metal salts, such as sodium salt, potassium salt, etc.; alkaline earth metal salts, such as calcium salt, magnesium salt, barium salt, aluminum salt, and the like. Non-limiting examples of salts formed with inorganic acids include, but are not limited to, salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like. Non-limiting examples of salts formed with organic acids include, but are not limited to, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzene Salts formed by sulfonic acid, p-toluenesulfonic acid, etc.

The carrier mentioned refers to the conventional carriers in the pharmaceutical field, such as diluents, excipients such as water, etc.; binders such as cellulose derivatives, gelatin, polyvinylpyrrolidone, etc.; fillers such as starch, etc.; disintegrating agents such as carbonic acid Calcium, sodium bicarbonate; in addition, other auxiliary agents such as flavoring and sweetening agents can also be added to the composition.

Various dosage forms of the composition of the present invention can be prepared by conventional methods in the medical field, and the content of the active ingredient is 0.1%-99.5% (weight ratio).

The dosage of the present invention can be changed according to the route of administration, the age, weight of the patient, the type and severity of the disease being treated, etc. The daily dosage is 0.001-30 mg/kg body weight (oral) or 0.005-30 mg/kg body weight ( injection).

Compared with the prior art, the pyrrolopyrimidine compounds, their stereoisomers and their pharmaceutically acceptable salts provided by the present invention have better inhibitory activity on two-sided god kinases, and their selectivity for JAK2 inhibitory targets is significant It is superior to existing compounds, and the preferred compounds of the present invention exhibit good pharmacokinetic properties, and have the potential to be developed as selective JAK2 inhibitors.

Detailed ways

The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Reference example 1: The synthetic route of fragment IB1.

Steps:

Add NaH (3.55g, 2eq, 60%) to DMF (150mL), cool to 0°C, drop cyanomethyl diethyl phosphite (15g, 2eq) into the above reaction solution, stir for 1 hour, and slowly rise to At room temperature, a solution of IA1 (10 g, 44.4 mol) in DMF (50 mL) was added dropwise, and stirring was continued for 12 hours. The reaction solution was poured into water, extracted with ethyl acetate, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Column chromatography with petroleum ether/ethyl acetate (10/1) yielded 9 g of colorless oily product IB1 with a yield of 76%. MS: 249.0[M+H] ⁺ , ¹ H NMR (400MHz, CDCl ₃ ) δ: 5.26 (s, 1H), 3.64-3.47 (m, 2H), 3.21-3.00 (m, 2H), 2.98-2.63 ( m, 4H), 2.56 (d, J = 16.0 Hz, 1H), 2.41 (d, J = 16.0 Hz, 1H), 1.44 (s, 9H).

Reference example 2-6: Fragment IB2～IB6.

With reference to the synthesis method of the operation steps in Reference Example 1, the reference examples in Table 2 below were synthesized:

Reference Example 7: Synthetic route of fragment IC1.

Operation steps: Step 1, the synthesis of IC1.

At room temperature, compound IB1 (9.0 g, 36.2 mol) and 2 (9.0 g, 28.5 mol) were added to acetonitrile (150 mL). DBU (5.6g, 37.1mol) was slowly dropped into the reaction system, the temperature was raised to 65°C, and the reaction was stirred for 24h. The reaction solution was quenched with water, extracted with ethyl acetate, separated, dried, filtered, and concentrated to obtain a crude oil. The crude oil was subjected to dichloromethane/methanol (20/1) column chromatography to obtain the colorless oily product IC1 (9.5 g, yield 59%). MS: 564.8[M+H] ⁺ , ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 8.78(s,0.55H), 8.73(d,J=3.2Hz,1H), 8.72(s,0.57H) ,8.38(d,J=7.2Hz,1H),7.77-7.71(m,1H),7.15(dd,J=7.2,3.7Hz,1H), 5.61(s,2H), 4.43(s,2H), 3.58-3.44 (m, 2H), 3.40-3.33 (m, 2H), 3.27-3.09 (m, 2H), 3.05-2.95 (m, 1H), 2.95-2.88 (m, 1H), 2.67-2.55 (m ,1H),2.48-2.42(m,1H),2.37-2.25(m,1H),1.84(dd,J=13.6,8.0Hz,1H),1.38(s,9H),0.88-0.71(m,2H ), -0.13(s,9H).

Step 2. Synthesis of ID1.

Compound IC1 (9.0 g, 16.0 mol) was added to 20% TFA dichloromethane solution (90 mL) and stirred at room temperature for 3 hours. Cool to 0°C, add saturated potassium bicarbonate solution, and adjust the pH to 8. Dichloromethane extraction, drying, and concentration to obtain crude oil. The crude oil was subjected to dichloromethane/methanol (20/1) column chromatography to obtain product ID1 (4.5 g, yield 61%). MS: 464.6[M+H]+, 1H NMR(400MHz,DMSO-d6)δ:8.89(s,1H),8.72(br,1H),8.61(s,1H),8.31(s,1H),7.45 (d, J = 4Hz, 1H), 6.86 (d, J = 4Hz, 1H), 5.69 (s, 2H), 3.54 (t, J = 12Hz, 2H), 2.60-2.30 (m, 6H), 2.30- 2.10 (m, 6H), 0.79 (t, J = 12 Hz, 2H), -0.06 (s, 9H). Reference example 8-12: Fragment ID2～ID6.

With reference to the synthesis method of Step 1 and Step 2 of Reference Example 7, the reference examples in Table 3 below were synthesized:

Example 1: I-1 and I-2

synthetic route:

Steps:

Compound ID1 (2.5g, 5.4mol) and triethylamine (2.3mL, 2eq) were sequentially dissolved in dichloromethane (50mL), the temperature was reduced to 0°C, and ethylsulfonyl chloride (1.38g, 10.7mol) was slowly added dropwise with stirring , Naturally rise to room temperature, stir and react for 2 hours. The reaction solution was washed with water (30 mL), saturated brine (30 mL), separated, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude intermediate IE1. This intermediate was added to 25% TFA/dichloromethane solution (16 mL), stirred at room temperature for more than 15 hours, concentrated under reduced pressure to remove the solvent and TFA, 15% ammonia-methanol solution (40 mL) was added, and stirred at room temperature for 3 hours. Concentrate to dryness. The residue was extracted with dichloromethane (50 mL)/water (30 mL), the organic layer was washed with saturated brine (30 mL), separated, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a crude mixture of 1-1 and 1-2. The mixture was prepared and separated by Pre-HPLC to obtain I-1 (550 mg, yield 24%) and I-2 (300 mg, yield 13%).

I-1: MS: 426.5 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ: 12.12 (br, 1H), 8.76 (s, 1H), 8.69 (s, 1H), 8.39 (s, 1H), 7.60 (d, J = 3.6 Hz, 1H), 7.05 (d, J = 3.6 Hz, 1H), 3.17 (q, J = 9.6 Hz, 2H), 3.10-3.00 (m, 8H), 2.63 (m, 2H), 1.79 (m, 2H), 1.10 (t, J=9.2 Hz, 3H).

I-2: MS: 426.5 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 12.11 (br, 1H), 8.91 (s, 1H), 8.68 (s, 1H), 8.41 (s, 1H), 7.61 (d, J = 3.6 Hz, 1H), 7.07 (d, J = 3.6 Hz, 1H), 3.24 (q, J = 9.2 Hz, 2H), 3.12-3.00 (m, 8H), 2.61 (m, 2H), 1.81 (m, 2H), 1.15 (t, J=9.2 Hz, 3H).

With reference to the synthesis method in Example 1, the compound of each example in Table 4 below was synthesized:

The NMR and MS data of each example compound are summarized in Table 5 below:

Biological test

Test example 1, JAK1, JAK2, JAK3, TYK2 activity test

Compound preparation:

The compound was dissolved in 100% DMSO, prepared as a 10mM stock solution, and frozen at -20°C.

Kinase reaction process:

(1) Prepare 1×Kinase buffer.

(2) Preparation of compound concentration gradient: The initial concentration of the test compound is 500 nM, which is diluted to a 100% DMSO solution of 100 times the final concentration in a 384 source plate, and the compound is diluted 3 times with Precision, 12 concentrations. Use a dispenser Echo 550 to transfer 250 nL 100 times the final concentration of the compound to the target plate OptiPlate-384F.

(3) Prepare a kinase solution of 2.5 times the final concentration with 1×Kinase buffer.

(4) Add 10 μL of 2.5 times the final concentration of kinase solution to the compound well and the positive control well; add 10 μL of 1×Kinase buffer to the negative control well.

(5) Centrifuge at 1000 rpm for 30 seconds, shake and mix the reaction plate, and incubate at room temperature for 10 minutes.

(6) Use 1×Kinase buffer to prepare a mixed solution of 5/3 times the final concentration of ATP and Kinase substrate.

(7) Add 15 μL of a mixed solution of 5/3 times the final concentration of ATP and substrate to initiate the reaction.

(8) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake and mix, and incubate at room temperature for the corresponding time.

(9) Add 30 μL of stop detection solution to stop the kinase reaction, centrifuge at 1000 rpm for 30 seconds, shake and mix.

(10) Use Caliper EZ Reader to read the conversion rate.

data analysis:

Calculation formula:

Among them: Conversion%_sample is the conversion rate reading of the sample; Conversion%_min: the average value of the negative control wells, which represents the conversion rate readings of the wells without enzyme activity; Conversion%_max: the average value of the positive control wells, represents the conversion rate readings of the wells without compound inhibition .

Fitting dose-effect curve:

Take the log value of the concentration as the X-axis, and the percentage inhibition rate on the Y-axis. Use the analysis software GraphPad Prism 5 log(inhibitor) vs.response-Variable slope to fit the dose-effect curve to obtain the IC50 value of each compound on the enzyme activity , Calculation formula:

Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))

The above experimental results are shown in Table 6.

Table 6. Test results of compound enzyme experiment:

Note: The above control samples and the compound of the present invention are all measured values under the same experimental conditions.

Conclusion: The compound of the present invention has better inhibitory activity and target selectivity against JAK2 than the positive controls Baricitinib and Ruxolitinib.

Test Example 2: Cell Proliferation Experiment

HEL92.1.7 cell proliferation experiment

Experimental steps:

(1) Paving

a. Digest and resuspend the cells and count them with an automatic cell counter.

b. Dilute the cell suspension to the desired density.

c. Pave 100ul cells in each well and incubate overnight at 37°C.

(2) Compound preparation

a. Prepare the compound into a diluted solution with a final concentration of 200 times.

b. Dilute the compound with culture medium to prepare a compound of 3 times the final concentration. Add 50ul compound to each well, and use the same volume of DMSO as the control, and incubate at 37°C and 5% CO _{2 for} 72 hours.

(3) Detection

a. Equilibrate the cell plate to room temperature.

试剂，振2分钟，静置10分钟，用EnVision检测。 b. Add 40μL Cell to each well

Reagent, shake for 2 minutes, stand for 10 minutes, and detect with EnVision.

data analysis:

(1) is calculated using GraphPad Prism 5 IC _50.

(2) %Inh=(Max signal-Compound signal)/(Max signal-Min signal) x 100.

(3) Max signal is a positive control well, with only the same volume of DMSO as the compound.

(4) Min signal is a negative control well with only medium.

TF-1 cell proliferation experiment

(1) Cell plating

a. Prepare complete medium.

b. Resuscitate the cells and cultivate the cells.

c. The cells are centrifuged, resuspended, counted, and plated. Place the culture plate in a CO ₂ incubator overnight.

(2) Preparation and addition of compounds

a. Use DMSO to formulate the compound into a 10mM stock stock solution, dilute 10mM to a working concentration, and gradually dilute by multiples to obtain compounds with multiple concentration gradients.

b. Pipette 0.5ul from the corresponding compound plate and add it to the cell culture plate cultured overnight.

c. Incubate for 72 hours in a 37°C incubator.

(3) Detection and analysis

a. Prepare CellTiter Glo assay reagents.

b. Add the detection reagents to the culture plate, mix well, stand still, and read the plate.

The inhibition rate formula is (1-(corresponding hole value-BLANK average value)/(DMSO control average value-BLANK average value))*100%)

) The curve fitting tool (XL fit) formula is Data Analysis:(XLfit software:Fit model:Dose response one site/

)

The above experimental results are shown in Table 7.

Table 7. Test results of cell proliferation experiment:

Note: The above control samples and the compound of the present invention are all measured values under the same experimental conditions.

Conclusion: The compound of the present invention has obvious proliferation inhibitory activity on HEL92.1.7 and TF-1, and the inhibitory activity is better than Baricitinib and Ruxolitinib.

Test Example 3. Pharmacokinetic test of the compound of the present invention

Taking SD rats as the test animals, the LC/MS/MS method was used to determine the rat's intragastric administration of Baricitinib, Ruxolitinib and the compounds of the preferred embodiments of the present invention, and then the drug concentration in plasma at different times was measured to study the compounds of the present invention in Pharmacokinetic characteristics in rats.

Source of SD rats: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

Administration method: single intragastric administration

Dosage and concentration: 25mg/kg; 1mg/mL

Preparation prescription: 0.5% methylcellulose

Sampling points: 5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h.

8.0计算实验结果。 Standard curve and quality control sample preparation and processing: Take an appropriate amount of stock solution and dilute with 50% acetonitrile water into a standard working solution of 0.04, 0.10, 0.20, 0.40, 1.00, 2.00, 4.00 μg/mL, 0.10, 1.00, 3.00 μg/mL Quality control working fluid. Take 47.5μL of blank rat plasma and add 2.50μL of standard curve working solution and quality control working solution respectively, and configure the standard curve with the concentration of analyte at 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00ng/mL Add 200μL of acetonitrile (containing the internal standard loratadine 5ng/mL) to the quality control samples with concentrations of 5.00, 50.00 and 150.00ng/mL, vortex for 3min, centrifuge at 15000rpm, 4℃ for 15min, and take the supernatant Liquid 100L was analyzed by LC-MS/MS. use

8.0 Calculate the experimental results.

The pharmacokinetic parameters of the preferred compounds of the present invention are shown in Table 8.

Table 8: Pharmacokinetic parameters of preferred compounds

Conclusion: The compounds of the examples of the present invention show good pharmacokinetic properties, and have obvious pharmacokinetic advantages compared with Baricitinib and Ruxolitinib.

The above descriptions are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the present invention. Within the scope of protection.

Claims (10)

A pyrrolopyrimidine compound represented by formula I, its stereoisomers or a pharmaceutically acceptable salt thereof,

among them: R ₁ is a C _1-6 alkyl group or a C _1-6 alkyl cyano group; the C _1-6 alkyl cyano group means that any hydrogen on the C _1-6 alkyl group is replaced by a cyano group; R ₂ and R ₃ are each independently hydrogen or C _1-6 alkyl; R ₄ is C _1-6 alkyl, -C(O)R ₅ , -C(O)NR ₆ R ₇ , -S(O) ₂ R ₈ or -S(O) ₂ NR ₉ R ₁₀ ; a is 0, 1 or 2; b is 0, 1 or 2; c is 0, 1 or 2; d is 0, 1 or 2; R ₅ is a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group or a C _1-6 alkyl cyano group. The C _1-6 alkyl cyano group refers to a C _1-6 alkane Any hydrogen on the group is replaced by a cyano group; R ₆ and R ₇ are each independently hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl or R ₆ , R ₇ and the connected N atom together form a 3-6 membered ring; R ₈ is C _1-6 alkyl or C _3-6 cycloalkyl; R ₉ and R ₁₀ are each independently hydrogen, C _{1 ~ 6} alkyl group, C _{3 ~ 6} cycloalkyl, or R _9, R ₁₀ together with the N atom form a 3- to 6-membered ring. The pyrrolopyrimidine compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 1, wherein said: R ₁ is a C _1-4 alkyl group or a C _1-4 alkyl cyano group; the C _1-4 alkyl cyano group means that any hydrogen on the C _1-4 alkyl group is replaced by a cyano group; R ₂ and R ₃ are each independently hydrogen, methyl or ethyl; R ₄ is C _1-6 alkyl, -C(O)R ₅ , -C(O)NR ₆ R ₇ , -S(O) ₂ R ₈ or -S(O) ₂ NR ₉ R ₁₀ ; a is 0, 1 or 2; b is 0 or 1; c is 0, 1 or 2; d is 1 or 2; R ₅ is a C _1-4 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group or a C _1-4 alkyl cyano group, and the C _1-4 alkyl cyano group refers to a C _1-4 alkane Any hydrogen on the group is replaced by a cyano group; R ₆ and R ₇ are each independently hydrogen, C _1-4 alkyl, C _3-6 cycloalkyl or R ₆ , R ₇ and the connected N atom together form a 3-6 membered alicyclic ring; R ₈ is C _1-5 alkyl or C _3-6 cycloalkyl; R ₉ and R ₁₀ are each independently hydrogen, C _{1 ~ 6} alkyl group, C _{3 ~ 6} cycloalkyl, or R _9, R ₁₀ together with the N atom form a 3- to 6-membered aliphatic ring. The pyrrolopyrimidine compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 1, wherein said: R ₁ is methyl, ethyl or

R ₂ and R ₃ are hydrogen; R ₄ is C _1-3 alkyl, -C(O)R ₅ , -C(O)NR ₆ R ₇ , -S(O) ₂ R ₈ or -S(O) ₂ NR ₉ R ₁₀ ; a is 0, 1 or 2; b is 0 or 1; c is 1 or 2; d is 1; R ₅ is C _1-3 alkyl, C _3-6 cycloalkyl,

R ₆ and R ₇ are each independently hydrogen, C _1-3 alkyl, C _3-6 cycloalkyl or R ₆ , R ₇ and the connected N atom together form a 5- to 6-membered alicyclic ring; R ₈ is a C _1-5 alkyl group or a C _4-5 cycloalkyl group; R ₉ and R ₁₀ are each independently hydrogen, C _1-3 alkyl, C _3-5 cycloalkyl, or R ₉ , R ₁₀ and the connected N atom together form a 5- to 6-membered alicyclic ring. The pyrrolopyrimidine compound, its stereoisomer, or a pharmaceutically acceptable salt thereof according to claim 1, wherein the stereoisomer is a stereoisomer formed by the C atom directly connected to R ₁ Construct. The pyrrolopyrimidine compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 1, wherein the pyrrolopyrimidine compound is as described in formula II:

The definition of each group is as described in claim 1. The pyrrolopyrimidine compound, its stereoisomer or its pharmaceutically acceptable salt according to claim 1, characterized in that it is selected from the following compounds:

A synthetic method for preparing the pyrrolopyrimidine compound according to any one of claims 1 to 6, characterized in that it comprises the following steps:

(1) Compounds of general formula IA are prepared by Wittig reaction to compounds of general formula IB; (2) Compound IC of general formula is prepared by catalyzed coupling reaction between IB and commercial compound 2; (3) IC is deprotected and condensation reaction is used to prepare IE; (4) Compound IE is deprotected to obtain the final product I; In the above reaction formula, the P1 group is a protecting group for N, and the specific group can be selected from C _1-6 alkoxycarbonyl; the X group is an activating group, and the specific group can be selected from halogen, C _1-3 alkoxy Group or C _1-3 alkanesulfonic acid group. The use of the pyrrolopyrimidine compound, its stereoisomers, and pharmaceutically acceptable salts according to any one of claims 1 to 6 in the preparation of drugs for preventing or treating JAK-related diseases. The use according to claim 8, characterized in that the JAK-related diseases include organ transplant rejection, lupus, multiple sclerosis, rheumatoid arthritis, juvenile arthritis, psoriasis, ulcerative colitis, gram Rohn’s disease, autoimmune thyroid disease, psoriasis, itching, atopic dermatitis, asthma, rhinitis, hepatitis B, hepatitis C, varicella-zoster virus, type I diabetes and diabetic complications, Al Zheimer's disease, dry eye, myelofibrosis, thrombocytosis, polycythemia, leukemia, multiple myeloma, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer , Thyroid cancer, glioblastoma, melanoma, lymphoma, leukemia, skin T-cell lymphoma or skin T-cell lymphoma. A composition comprising a therapeutically effective amount of the pyrrolopyrimidine compound according to any one of claims 1 to 6, its stereoisomer or its pharmaceutically acceptable salt, and a pharmaceutically acceptable a.